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Biomedical subjects

S J Elliott

Publications and source records attributed to S J Elliott.

At least 19 recordsLinked to original sources
PubMed1998

The particulate methane monooxygenase from methylococcus capsulatus (Bath) is a novel copper-containing three-subunit enzyme. Isolation and characterization.

The particulate methane monooxygenase (pMMO) is known to be very difficult to study mainly due to its unusual activity instability in vitro. By cultivating Methylococcus capsulatus (Bath) under methane stress conditions and high copper levels in the growth medium, membranes highly enriched in the pMMO with exceptionally stable activity can be isolated from these cells. Purified and active pMMO can be subsequently obtained from these membrane preparations using protocols in which an excess of reductants and anaerobic conditions were maintained during membrane solubilization by dodecyl beta-D-maltoside and purification by chromatography. The pMMO was found to be the major constituent in these membranes, constituting 60-80% of total membrane proteins. The dominant species of the pMMO was found to consist of three subunits, alpha, beta, and gamma, with an apparent molecular mass of 45, 26, and 23 kDa, respectively. A second species of the pMMO, a proteolytically processed version of the enzyme, was found to be composed of three subunits, alpha', beta, and gamma, with an apparent molecular mass of 35, 26, and 23 kDa, respectively. The alpha and alpha' subunits from these two forms of the pMMO contain identical N-terminal sequences. The gamma subunit, however, exhibits variation in its N-terminal sequence. The pMMO is a copper-containing protein only and shows a requirement for Cu(I) ions. Approximately 12-15 Cu ions per 94-kDa monomeric unit were observed. The pMMO is sensitive to dioxygen tension. On the basis of dioxygen sensitivity, three kinetically distinct forms of the enzyme can be distinguished. A slow but air-stable form, which is converted into a "pulsed" state upon direct exposure to atmospheric oxygen pressure, is considered as type I pMMO. This form was the subject of our pMMO isolation effort. Other forms (types II and III) are deactivated to various extents upon exposure to atmospheric dioxygen pressure. Under inactivating conditions, these unstable forms release protons to the buffer (approximately 10 H+/94-kDa monomeric unit) and eventually become completely inactive.

Amino Acid Sequence↗
PubMed1998

Characterization of the roles of hemolysin and other toxins in enteropathy caused by alpha-hemolytic Escherichia coli linked to human diarrhea.

Escherichia coli strains producing alpha-hemolysin have been associated with diarrhea in several studies, but it has not been clearly demonstrated that these strains are enteropathogens or that alpha-hemolysin is an enteric virulence factor. Such strains are generally regarded as avirulent commensals. We examined a collection of diarrhea-associated hemolytic E. coli (DHEC) strains for virulence factors. No strain produced classic enterotoxins, but they all produced an alpha-hemolysin that was indistinguishable from that of uropathogenic E. coli strains. DHEC strains also produced other toxins including cytotoxic necrotizing factor 1 (CNF1) and novel toxins, including a cell-detaching cytotoxin and a toxin that causes HeLa cell elongation. DHEC strains were enteropathogenic in the RITARD (reversible intestinal tie adult rabbit diarrhea) model of diarrhea, causing characteristic enteropathies, including inflammation, necrosis, and colonic cell hyperplasia in both small and large intestines. Alpha-hemolysin appeared to be a major virulence factor in this model since it conferred virulence to nonpathogenic E. coli strains. Other virulence factors also appear to be contributing to virulence. These findings support the epidemiologic link to diarrhea and suggest that further research into the role of DHEC and alpha-hemolysin in enteric disease is warranted.

Animals↗
PubMed1998

Peroxynitrite is a contractile agonist of cerebral artery smooth muscle cells.

On reperfusion of ischemic tissue, a prolonged phase of vasoconstriction occurs, the mechanism of which is poorly understood. However, it is known that peroxynitrite (ONOO-) is formed during reperfusion. In this study the contractile properties of ONOO- were investigated in Wistar rat middle cerebral arteries. The effects of ONOO- on vessel diameter were dose dependent. Low-dose ONOO- (10 microM) caused vessels to constrict by 15%. At an intermediate concentration of 25 microM, the effect of ONOO- was variable, whereas at the highest concentration (100 microM), vessels underwent persistent dilation and became insensitive to the endogenous vasoconstrictor 5-hydroxytryptamine. At the single cell level, ONOO- caused cerebral artery smooth muscle cells to contract. Reduced, but not oxidized, glutathione completely inhibited the contractile action of ONOO- on single cells. Vehicle and decomposed ONOO- each had minimal effect on cell length. These data show that ONOO- is a contractile agonist of middle cerebral arteries, at the single cell and whole vessel levels, suggesting that formation of ONOO- may contribute mechanistically to ischemic brain injury during stroke. Moreover, relatively high concentrations of ONOO- result in vascular paralysis.

Animals↗
PubMed1997

Role of type 1 fimbriae in EPEC infections.

Several fimbriae have been implicated as potentially important in EPEC adhesion and pathogenesis. EPEC strain E2348/69 produced only bundle forming pili and type 1 fimbriae, and did not produce other accesory adhesins identified in EPEC strain B171. Cloning and mutagenesis of these EPEC fim genes indicated that type 1 pili had no affect on levels or patterns of adhesion to cultured human cells.

Animals↗
PubMed1997

Redox control of ion channel activity in vascular endothelial cells by glutathione.

Oxidized glutathione (GSSG) is endogenously formed within vascular endothelial cells. The bioactivity of GSSG results in the oxidation of protein thiol groups, leading to changes in protein structure-function relationships. When ion channel protein thiols are the target of oxidation by GSSG, important changes in channel conductance, activity, and gating occur. In this review, we focus on two endothelial cell ion channels, the activities of which influence vascular cell signaling and the nitric oxide signaling pathway. The first channel is the GSSG-operated cation channel that depolarizes the endothelial cell, leading to inhibition of capacitative Ca2+ entry. The second channel is the inositol 1,4,5-triphosphate (IP3)-operated Ca2+ channel that is responsible for the agonist-stimulated release of Ca2+ from IP3-sensitive endoplasmic reticulum. GSSG acts to deplete IP3-sensitive Ca2+ stores, thereby attenuating the intracellular Ca2+ response to agonist stimulation. Together, these effects indicate that glutathione, which is formed endogenously within the cell, is a key physiological modulator of endothelial cell signaling.

Animals↗
PubMed1996

Oxidized glutathione mediates cation channel activation in calf vascular endothelial cells during oxidant stress.

1. The oxidant, tert-butylhydroperoxide (tBuOOH) depolarizes calf pulmonary artery endothelial cells by activating a non-selective cation channel. To identify the molecular mediator of channel activation during oxidant stress, the patch-clamp technique was used to compare tBuOOH-induced changes in membrane potential and channel activity with those induced by oxidized glutathione (GSSG), a cytosolic product of oxidant metabolism. 2. When recording pipettes contained GSSG (2 mM), whole-cell zero-current potential measured immediately following pipette break-in was not different from control values (-57 mV). However, within 20 min of break-in, zero-current potential was depolarized to -7 mV. The time course of depolarization was dependent on the concentration of GSSG and was accelerated by inhibition of GSSG metabolism. 3. In excised membrane patches, channels were activated by internal GSSG, but not by internal tBuOOH, reduced glutathione (GSH), or external GSSG. Channels were equal in size (28 pS) and in ionic selectivity to those activated by incubation of intact cells with tBuOOH. As little as 20 microM GSSG was sufficient to maximally activate channels. However, the time course of channel activation was concentration dependent between 20 microM and 2 mM GSSG. 4. Channel activation by GSSG was reversed by GSH and by increasing the [GSH]:[GSSG] ratio. Likewise, channel activation by pre-incubation of intact cells with tBuOOH was reversed by GSH applied after patch excision. 5. These results strongly suggest that GSSG is an endogenous intracellular mediator of channel activation and depolarization during oxidant stress.

Animals↗
PubMed1996

Oxidant stress activates a non-selective cation channel responsible for membrane depolarization in calf vascular endothelial cells.

1. In vascular endothelial cells, oxidant stress increases cell Na+ content and inhibits the agonist-stimulated influx of external Ca2+. Further, oxidant stress increases uptake of Ca2+ into otherwise quiescent endothelial cells. To determine the mechanism responsible for altered Na+ and Ca2+ homeostasis, the present study examined the effect of oxidant stress on ionic current and channel activity in calf pulmonary artery endothelial cells. 2. Voltage-clamped control cells had a zero-current potential of -60 mV. Incubation of cells with the oxidant tert-butylhydroperoxide (tBuOOH; 0.4 mM, 1 h) caused depolarization to -4 mV and activation of ionic current equally selective for Na+ and K+. 3. Cell-attached membrane patches made on tBuOOH-treated cells contained ion channels that had a bidirectional conductance of 30 pS and that were not present in patches from control cells. Inside-out patches excised from oxidant-treated cells showed the channel to be equally selective for Na+ and K+ and to allow inward Ca2+ current. 4. Oxidant-activated channels were observed to display two gating modalities that were further evident during analysis of single-channel open probability. Neither modality was significantly affected by altering internal [Ca2+] (1 microM-10 nM). 5. Activation of non-selective channels provides a possible mechanism by which oxidants may increase endothelial cell Na+ content. Channel permeability to Ca2+ may account in part for the elevation of cytosolic free [Ca2+] that occurs in oxidant-treated cells. 6. Channel activation is associated with membrane depolarization, a mechanism that may contribute to oxidant inhibition of the agonist-stimulated Ca2+ influx pathway.

Animals↗
PubMed1996

Peroxynitrite modulates receptor-activated Ca2+ signaling in vascular endothelial cells.

Peroxynitrite (ONOO-) is formed from superoxide (O2-) and .NO. We have previously reported that O2- does not alter endothelial cell Ca2+ signaling. To test whether .NO alters Ca2+ signaling, cells were incubated with the .NO donor, spermine NONOate. Neither spermine NONOate nor S-nitroso-N-acetyl penicillamine (SNAP) altered bradykinin-stimulated Ca2+ signaling. By contrast, 3-morpholinosydnonimine (SIN-1), which generates ONOO- by releasing O2- and .NO essentially in a simultaneous manner, significantly inhibited signaling. Initially, the inhibitory effect of 1 mM SIN-1 was selective toward agonist-stimulated influx of external Ca2+. At later time points, SIN-1 additionally depleted internal stores of releasable Ca2+. When cells were coincubated with SIN-1 plus superoxide dismutase, a technique designed to scavenge O2- and convert SIN-1 to purely an .NO-donor compound, Ca2+ signaling was identical to control. SIN-1C, the inactive metabolite of SIN-1, had no effect on [Ca2+]i. This study demonstrates that exogenously generated ONOO- modulates endothelial cell Ca2+ signaling, suggesting that ONOO- is of biological relevance to vasoregulation.

Animals↗
PubMed1995

Oxidized glutathione decreases luminal Ca2+ content of the endothelial cell ins(1,4,5)P3-sensitive Ca2+ store.

The model oxidant, t-butyl hydroperoxide (t-buOOH), inhibits Ins(1,4,5)P3-dependent Ca2+ signalling in calf pulmonary artery endothelial cells. Metabolism of t-buOOH within the cytosol is coupled to the oxidation of glutathione. In this study, we investigated whether oxidized glutathione (GSSG) is the intracellular moiety responsible for mediating the effects of t-buOOH on Ca2+ signalling. The increase in cytosolic [Ca2+] stimulated by application of 2,5-di-t-butylhydroquinone (BHQ) was used to estimate the luminal Ca2+ content of the Ins(1,4,5)P3-sensitive store in intact cells. Luminal Ca2+ content was unaffected by t-buOOH (0.4 mM, 0-3 h) unless intracellular GSSG content was concomitantly elevated. The effect was specific for increased GSSG and was not replicated by depletion of GSH. These results suggest that cytosolic GSSG, produced endogenously within the endothelial cell, decreases the luminal Ca2+ content of Ins(1,4,5)P3-sensitive Ca2+ stores. Depletion of internal Ca2+ stores by GSSG may represent a key mechanism by which some forms of oxidant stress inhibit signal transduction in vascular tissue. At the plasma membrane, t-buOOH is known to inhibit the capacitative Ca2+ influx pathway. Increased intracellular GSSG potentiated the inhibitory effect of t-buOOH on Ca2+ influx, thereby providing the first evidence that activity of the capacitative Ca2+ influx channel is sensitive to thiol reagents formed endogenously within the cell.

Animals↗
PubMed1995

Virulence factors associated with strains of Escherichia coli from cases of sudden infant death syndrome (SIDS).

Strains of Escherichia coli isolated from cases of Sudden Infant Death Syndrome, healthy infants and infants that died of other causes were subjected to a series of tests with particular reference to serotyping, toxigenicity and adherence factors. E. coli from SIDS infants tended to have a low hydrophobicity and high toxigenicity, compared to those from healthy infants, while no notable differences in haemagglutination patterns were observed between these two groups of strains.

Animals↗
PubMed1995

Psychosocial stress, women and heart health: a critical review.

This paper reports the results of a systematic critical appraisal of the research literature designed to determine the balance of evidence for the role of psychosocial stress as a risk factor in women's coronary disease. The study is placed within a larger research programme which addresses geographic variation in heart disease among women. The scope of the research is based on the burden of illness that coronary disease represents, existing geographic variation in morbidity and mortality, the role of psychosocial stress as a potential risk factor for women given changing gender roles, and the relative lack of attention paid to both the etiology and epidemiology of women's coronary disease in the research literature. In fact, there is very little original research, the balance of which provides equivocal evidence of a link between psychosocial stress and coronary disease in women but enough to suggest a need for further etiologic research. This need is substantiated by the appearance of a perception among the general population that 'stress' 'causes' heart disease. In addition, it would seem that 'stress' and 'heart disease' are major health concerns for women. Given the dichotomy between actual and perceived etiologic links, there perhaps needs to be an adjustment made with respect to research focus which addresses the role of perceived environmental stress as well as the individual in defining health and well-being. That is, the relationship between psychosocial stress and heart disease may depend upon the meaning of the situation to the individual and the way she perceives her life situation. Medical geographers are well-placed to address these research issues using a combination of qualitative and quantitative approaches.

Coronary Disease↗
PubMed1995

Oxidant stress and endothelial membrane transport.

The endothelium modulates vascular tone, vasoreactivity, and permeability in response to agonist-stimulation. Much of the pathophysiology of oxidant-induced vascular injury can be attributed to endothelial cell dysfunction. In the past several years, the effects of oxidant stress on agonist-stimulated Ca(2+)-channels have been described. More recently, the effects of oxidant stress on several other endothelial membrane-transport systems have been elucidated. It now appears that inhibition of the agonist-stimulated Ca2+ channel is due at least in part to membrane depolarization via oxidant-activation of a Na(+)-permeable, nonselective cation channel. In this review, the effects of oxidant stress on ion transport through the agonist-stimulated Ca2+ influx channel, Na+ and K+ channels, Na+/K(+)-ATPase, Ca(2+)-ATPase, and the Na+/K+/2Cl- cotransporter are discussed. The interrelated effects of oxidant stress on these endothelial membrane transport pathways are considered, and the net effect on Ca2+ signaling is described.

Animals↗
PubMed1995

Reductant substrate for glutathione peroxidase modulates oxidant inhibition of Ca2+ signaling in endothelial cells.

Oxidant stress mediated by tert-butyl hydroperoxide (t-BOOH) inhibits agonist-stimulated Ca2+ entry and internal store Ca2+ release in cultured endothelial cells. The role of intracellular glutathione in modulating the effects of oxidant stress on Ca2+ signaling was determined in cells preincubated with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase, or 1-chloro-2,4-dinitrobenzene (CDNB), a cosubstrate for glutathione-S-transferase. BSO and CDNB decreased endothelial cell glutathione content by 85 and 97%, respectively (control glutathione, 21.5 +/- 2.3 nmol/mg protein). Each agent accelerated the time-dependent effects of t-BOOH on Ca2+ signaling in fura 2-loaded cells and potentiated the inhibition of bradykinin-stimulated 45Ca2+ efflux induced by t-BOOH. These results indicate that decreased availability of reduced glutathione, the primary cosubstrate for glutathione peroxidase, potentiates the effect of hydroperoxide oxidant stress on receptor-operated Ca2+ entry across the plasmalemma and Ca2+ release from internal stores. The present findings suggest that intracellular glutathione availability and/or glutathione redox cycle activity are critically important modulators of oxidant inhibition of Ca(2+)-dependent signal transduction.

Animals↗
PubMed1995

The H2O2-generating enzyme, xanthine oxidase, decreases luminal Ca2+ content of the IP3-sensitive Ca2+ store in vascular endothelial cells.

OBJECTIVE: Xanthine oxidase inhibits agonist-stimulated Ca2+ signaling in calf pulmonary artery endothelial cells by an H2O2-dependent mechanism. We investigated the effect of xanthine oxidase on luminal Ca2+ content of the inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ store. METHODS: Luminal Ca2+ content was estimated from the net release of Ca2+ activated by 2,5-di-t-butylhydroquinone (BHQ), an inhibitor of microsomal Ca2+ pumps. RESULTS: Initially, xanthine oxidase depleted the IP3-sensitive Ca2+ store of releasable Ca2+, but with more prolonged incubation, the enzyme also depleted non-IP3-sensitive stores. In addition, xanthine oxidase inhibited capacitative Ca2+ influx. Similar results were observed when thapsigargin was substituted for BHQ. CONCLUSIONS: Depletion of luminal Ca2+ content within the IP3-sensitive Ca2+ store contributes to xanthine oxidase inhibition of Ca2+ signaling in vascular endothelial cells.

Animals↗